CN102597774A - Binding assay with multiple magnetically labelled tracer binding agents - Google Patents
Binding assay with multiple magnetically labelled tracer binding agents Download PDFInfo
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- CN102597774A CN102597774A CN201080047534XA CN201080047534A CN102597774A CN 102597774 A CN102597774 A CN 102597774A CN 201080047534X A CN201080047534X A CN 201080047534XA CN 201080047534 A CN201080047534 A CN 201080047534A CN 102597774 A CN102597774 A CN 102597774A
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/72—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables
- G01N27/74—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids
- G01N27/745—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids for detecting magnetic beads used in biochemical assays
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Abstract
The present invention relates to a cartridge (1) for the detection of an analyte (2) in a binding assay with magnetic labels (3), the cartridge comprising at least one capture binding agent (4) against a binding site on the analyte and comprising at least two magnetically labelled tracer binding agents (51 and 52) against further different binding sites on the analyte, characterised in that the cartridge comprises at least two regions (61 and 62) wherein a first region (61) comprises the at least one capture binding agent (4) and a first magnetically labelled tracer binding agent (51) and wherein a second region (62) comprises at least one capture binding agent (4) and a second magnetically labelled tracer binding agent (52). Herein the first region (61) does not comprise a magnetically labelled tracer binding agent other than the first magnetically labelled tracer bind agent (51) and wherein the second region (62) does not comprise a magnetically labelled tracer binding agent other than the second magnetically labelled tracer binding agent (52).
Description
The present invention relates to utilize the for example combination of the magnetic mark binding reagents of antibody to measure.
Sandwich immunoassay is the well-known method that is used for the check and analysis thing; Wherein common, be attached to the substrate (capture antibody) in the reaction chamber and be attached to detectable (tracer antibody) to the SA of same analyte (soluble or suspend) there to the first antibody of this analyte.This detectable label comprises color development label (for example, fluorescent marker) and magnetic-particle.
In this sandwich immunoassay,, one of antibody can not detect this analyte when not being attached to analyte.This can be for example by the change (for example, (going) phosphorylation) of epi-position on the degraded (for example, proteolysis) of analyte, the analyte or with the compound formation that prevents epi-position and other protein of antibodies on the analyte and cause.Above phenomenon possibly cause the false negative result in the disease marker detection.
Troponin is the generally acknowledged mark that is used for various heart diseases.Yet this protein has formed different subunits and after collecting blood sample, has been easy to degraded.In order to address this problem, developed the mensuration of wherein using two kinds of different tracer antibodies [people such as Eriksson. (2006) Clin.Chem.51,839-847; European patent application EP P1473567] [Fig. 1].In the prior art of this type is measured, come this tracer antibody of mark with little fluorescence organic molecule.
Utilize the magnetic mark thing to carry out this mensuration and produced unforeseen problem.Because with the caused steric hindrance of relative large scale of the size compared magnetic-particle of protein analyte, cause making the combining of two kinds of magnetic mark tracer antibodies and analyte (so-called bridge joint) the very difficult capture antibody [Fig. 2] that further is attached to for this analyte.
Need further to regulate the sandwich assay of utilizing different magnetic mark tracer antibodies with execution.
In independence and dependent claims, stated of the present invention specific and preferred aspect.Characteristic from dependent claims can combine with the characteristic of independent claims and the characteristic of other dependent claims as one sees fit, and not only clearly statement in claim.
The present invention prevents false negative result, in false negative result, will can not detect the disease marker that in sample, exists, and ill people or people with ill risk will be considered to healthy.
One aspect of the present invention relates to a kind of box body (1) that is used for utilizing in combination mensuration magnetic mark thing (3) check and analysis thing (2); Said box body comprises at least a binding reagents (4) of catching to the binding site on the said analyte; And comprise at least two kinds of magnetic mark spike binding reagents (51 and 52) to the other different binding site on the said analyte; It is characterized in that said box body comprises at least two zones (61 and 62); Wherein first area (61) comprise and saidly at least aly catch the binding reagents (4) and the first magnetic mark spike binding reagents (51), and wherein second area (62) comprises at least a binding reagents (4) and the second magnetic mark spike binding reagents (52) of catching.Said in this article first area (61) does not comprise the magnetic mark spike binding reagents of the non-said first magnetic mark spike binding reagents (51), and wherein said second area (62) does not comprise the magnetic mark spike binding reagents of the non-said second magnetic mark spike binding reagents (52).
Specific embodiment according to the box body of describing in this article; These comprise that two kinds to two different binding sites on the said analyte are caught binding reagents (41 and 42); Wherein said first area (61) comprises said first and catches binding reagents (41), and wherein said second area (62) comprises said second and catches binding reagents (42).
Other specific embodiments according to the box body of describing in this article; These comprise that two kinds to two different binding sites on the said analyte are caught binding reagents (41 and 42); Wherein said first area (61) comprises said first and catches binding reagents (41) and the said first spike binding reagents (51), and wherein said second area (62) comprises said second and catches binding reagents (42) and the said second spike binding reagents (52).
Other specific embodiments according to the box body of describing in this article; These comprise that two kinds to two different binding sites on the said analyte are caught binding reagents (41 and 42), and wherein said first area (61) and said second area (62) both comprise that said first catches binding reagents (41) and said second and catch binding reagents (42).
Still another specific embodiment according to the box body of describing in this article; These comprise that two kinds to two different binding sites on the said analyte are caught binding reagents (41 and 42); And comprise two kinds of magnetic mark spike binding reagents (51 and 52) to two other the different binding sites on the said analyte; Wherein said box body comprises two zones (61 and 62); Wherein first area (61) comprise that said first and second catch binding reagents (41 and 42) and the said first magnetic mark spike binding reagents (51), and wherein said second area (62) comprises that said first and second catch binding reagents (41 and 42) and the said second magnetic mark spike binding reagents (52).
According to other specific embodiments of the box body of describing in this article, binding reagents is an antibody.
According to other specific embodiments of the box body of describing in this article, the zone is a reaction chamber.
According to other specific embodiments of the box body of describing in this article, said analyte is a troponin, for example cardiac muscle troponin I (cTnI).
Another aspect of the present invention relates to a kind of magnetic mark spike binding reagents that uses with combining to measure the method that detects the analyte (2) in the sample, and said method comprises step:
-a) have at least two zones box body (2) of (61 and 62) is provided, wherein will be fixed on the surface of said zone (61 and 62) to the binding reagents (4) of catching of the binding site on the said analyte,
-b) compound of the said analyte and the first magnetic mark spike binding reagents being provided in said first area (61), the said first magnetic mark spike binding reagents is to the other binding site on the said analyte (51),
-c) compound of the said analyte and the second magnetic mark spike binding reagents (52) being provided in said second area (62), the said second magnetic mark spike binding reagents (52) is to the other again binding site on the said analyte,
-d) allow step b) and c) in said compound combine with the said binding reagents of catching in the said zone,
-e) detect step b) and c) in said compound to said combination of catching binding reagents,
Wherein in said method; Said first area (61) does not comprise the magnetic mark spike binding reagents of the non-said first magnetic mark spike binding reagents (51), and said second area (62) does not comprise the magnetic mark spike binding reagents of the non-said second magnetic mark spike binding reagents (52).
According to other specific embodiments of the method for describing in this article, comprise that in introducing the liquid of said analyte provides the spike binding reagents before in said zone.
According to other specific embodiments of the method for describing in this article, binding reagents is an antibody.
According to other specific embodiments of the method for describing in this article, the zone is a reaction chamber.
Further specific embodiment according to the method for describing in this article; Said analyte is a troponin; Cardiac muscle troponin I (cTnI) for example; Wherein tracer antibody (41) and capture antibody (51) are attached to the different epi-positions on the stabilized zone of cTnI, and wherein tracer antibody (42) and capture antibody (52) are attached to the different epi-positions on the unstable region of troponin.
Another aspect of the present invention relates to a kind of equipment of measuring that is used to use magnetic actuation to carry out to combine, and comprises box body described above.
Another aspect of the present invention relates to a kind of kit; At least three kinds of different binding reagents that comprise the different binding sites that are attached on the same analyte; Use the magnetic-particle mark at least two kinds in the wherein said binding reagents, and at least a without the magnetic-particle mark in the wherein said binding reagents.
According to the specific embodiment of the kit of describing in this article, kit comprises four kinds of different binding reagents of four different binding sites that are attached on the same analyte, uses the magnetic-particle mark for two kinds in wherein said four kinds of binding reagents.
According to another specific embodiment of the kit of describing in this article, said binding reagents is an antibody.
According to the specific embodiment of the kit of describing in this article, said analyte is a troponin.
Specific embodiment according to the kit of describing in this article; Said analyte is a cardiac muscle troponin I; And the said first unmarked antibody and the said first magnetic mark antibodies different epi-positions to the stabilized zone of cardiac muscle troponin I; And, the said second unmarked antibody and the said second magnetic mark antibodies different epi-positions to the unstable region of cardiac muscle troponin I.
Through following detailed description and combine mode by way of example to illustrate the accompanying drawing of the principle of the invention, of the present inventionly abovely will become obvious with other characteristics, characteristic and advantage.Be merely for example and provided this description, and do not limited the scope of the invention.The reference diagram of below quoting relates to accompanying drawing.
Fig. 1 shows the schematic overview of prior art immunoassays.A kind of capture antibody (4) combines with analyte (2) with two kinds of different tracer antibodies (51,52).Star (L) is represented fluorescent marker.
Fig. 2 shows the schematic overview when the steric hindrance that in the mensuration that Fig. 1 described, runs into during with the little fluorescent marker of big magnetic-particle (3) replacement.
Fig. 3 shows the exemplary embodiment according to box body of the present invention (1), and this box body has two zones (61,62), a kind of capture antibody (4) and two kinds of magnetic mark tracer antibodies (51 and 52).In vertical direction, upward go up this magnetic mark tracer antibody of lower-pilot (51 and 52) along axle Z in this article towards capture antibody (4) at respective regions (61) and (62).
Fig. 4 shows the exemplary embodiment according to box body of the present invention (1), and this box body has two reaction chambers (61,62), a kind of capture antibody (4) and adheres to the two kinds of tracer antibodies (51 and 52) with check and analysis thing (2) with magnetic-particle (3).
Fig. 5 shows the exemplary embodiment according to box body of the present invention (1), and this box body has two reaction chambers (61,62), two kinds of capture antibodies (41 and 42) and adheres to the two kinds of tracer antibodies (51 and 52) with check and analysis thing (2) with magnetic-particle (3).
Fig. 6 shows the exemplary embodiment according to box body of the present invention (1), and this box body has two reaction chambers (61,62), two kinds of capture antibodies (41 and 42) and adheres to the two kinds of tracer antibodies (51 and 52) with check and analysis thing (2) with magnetic-particle (3).
Fig. 7 shows the schematic structure (obtaining from Hytest catalogue (Hytest, Turku, Finland)) of TnC/Troponin I compound.
In different figure, same Reference numeral relates to identical or similar key element.Though it is the specific embodiment of antibody that figure provides binding reagents, key element (4), (41), (42), (5) likewise relate to other binding reagents that further detail as in the present invention.
To scheme to describe the present invention about specific embodiment and with reference to some, but the invention is not restricted to this specific embodiment and figure but be only limited to claim.Any Reference numeral in the claim should not be interpreted as limited field.Described figure is schematic rather than restrictive.In the drawings, the size of some key elements possibly be exaggeration and draw in proportion for illustration purposes.When in this instructions and claim, using a technical term " comprising ", it does not get rid of other key elements or step.Mentioning odd number, " one " perhaps " one ", " said " and use indefinite or during definite article for example, unless explicit state in addition, this comprises the plural number of that odd number.
In addition, term first, second, third in instructions and claim or the like is used for region class like key element, and must description order or chronological order.The term that will be appreciated that use like this is interchangeable under suitable situation, and the embodiments of the invention of describing in this article can be with other sequential workings in this paper, describing or illustrating.
Following term or definition just are provided for and help to understand the present invention.These definition should not be interpreted as has the scope of understanding less than those of ordinary skills.
" zone " in context of the present invention relates to the part of reaction chamber or relates to the entire reaction chamber, wherein during analyte/spike binding reagents compound and the interaction of catching binding reagents, has magnetic mark spike binding reagents.
" reaction chamber " in context of the present invention relates to the part of equipment or box body, spike binding reagents (being typically antibody), analyte wherein takes place and catch the compound formation between the binding reagents (being typically antibody).The part of the fixed trapped binding reagents of reaction chamber is called as " reaction surface ".This reaction chamber typically is the part of the passage between sample application district (inlet) and the outlet, widened section for example capillaceous.Usually, reaction chamber has at least one flat surfaces of fixed trapped binding reagents.Except the formation of above compound, this reaction chamber mainly also is used to detect the formation of this compound and also play " detection chambers ".When using optical devices to detect, this reaction chamber comprises at least one light transmission part.For most commercial application, reaction chamber is integrated in the box body, and this box body is to be inserted into the unit that comprises in the equipment capillaceous, to send and to remove sample fluid, flush fluid and buffer fluid.This box body typically also comprises object, and for example dried reagent (lytic agent) and damping fluid and filtrator are to remove particle matter from sample.The box body that uses in the present invention comprises at least one reaction chamber with separated region or the reaction chamber of at least two separations.When use has reaction chamber of same district not, separate and mean and can not spike binding reagents (being typically antibody) be handled another district to this reaction chamber from a district of this reaction chamber.When use had reaction chamber of same district not, " separation " meaned and can not spike binding reagents (typical case is an antibody) be handled to other reaction chambers from a reaction chamber.
Yet reaction chamber can be used to carry out multiplexing mensuration, wherein will be placed on different reaction surface places to the different spike binding reagents of different analytes; And has collateral condition: when using a reaction chamber; Like different spike binding reagents (51,52) to the different binding sites on the same analyte described in the invention not in the same area, perhaps under the situation of using the differential responses chamber; This difference spike binding reagents (51,52) is not in same reaction chamber.
" sandwich assay " relates to well-known combination type; Wherein the antibody via first epi-position that is directed against analyte is fixed to the surface with analyte; And utilize the antibody of second epi-position that is directed against this analyte to detect this analyte, wherein this SA carries detectable label.
" binding reagents " relates to the group that combines with a part of specificity of analyte.Typical example be the antibody that combines with the epi-position of protein perhaps its fragment (Fab, Fab (2), scFv).This antibody typically is monoclonal antibody, though the polyclonal antibody that is prepared as to epi-position also is same being fit to.Other examples of binding reagents are adaptive son, nano antibody, the anti-transporter of affine body (affibodies anticalins); Be used to be attached to the carbohydrates conjugated protein of carbohydrate analysis thing, for example agglutinin; Be used to be attached to the carbohydrates of carbohydrates conjugated protein analyte; Be used to be attached to the oligonucleotides or the polynucleotide of DNA-or RNA-conjugated protein; Be attached to the organic molecule (for example zymolyte or enzyme inhibitor, the antagonist of part or acceptor) of protein analyte; Be attached to peptide, protein or its part (the for example part of polyprotein matter compound, perhaps protein ligands receptor complex) of other peptides or protein.
" analyte " in context of the present invention relates to the compound that can be detected via binding reagents is attached to the binding site on this analyte.This analyte typically is a protein, and wherein this binding site is the epi-position of said combination of proteins to antibody.But this protein analyte can be single polypeptide also can be the compound of homopolypeptide not.Therefore; Described in this article mensuration comprises the check to multi-subunit protein matter compound, and wherein for example the binding reagents of antibody is attached on the binding site that exists in the different subunits of this compound (being epi-position under the situation that antibody/protein combines).Other analytes comprise carbohydrates, compound organic molecule for example enzyme inhibitor, receptors bind reagent, hormone, medical compounds.
" magnetic mark thing " relates to the magnetic that comprises arbitrary shape (being typically sphere) or the particle of magnetisable material, and it can be handled by magnetic field.
The diameter that the magnetic-particle size that the typical case uses in immunoassays is had is between about 50 to 2000nm; Specifically between about 250 to 750nm; And more specifically be about 500nm; It possibly cause unforeseen problem in the combination that utilizes two kinds or more kinds of spike binding reagents is measured, this spike binding reagents is for example by the antibody of magnetic-particle mark.In sandwich assay, tracer antibody at first combines with analyte, then the compound between this analyte and the tracer antibody is contacted with the capture antibody that is fixed.In based on the mensuration of magnetic-particle, also follow this program.Tracer antibody stir to be handled to allow combining of tracer antibody and analyte by magnetic in this article.After this, the capture antibody of the compound between manipulation analyte and the tracer antibody on detecting.The tracer antibody and the capture antibody that then, will not have an analyte through applying a magnetic field remove.The detection of analyte occurs in the wherein surface of fixed trapped antibody.
Quantity in conjunction with particle is relevant with the amount of the analyte molecule that in sample, exists.
Use two kinds of different spike binding reagents, for example every kind all by the antibody of magnetic-particle mark, obtains analyte molecule and be positioned in two kinds of compounds between the magnetic-particle.The size of this magnetic-particle possibly stop analyte and catch combining of binding reagents.Under the situation that this combination does not have to take place, analyte-spike binding reagents compound also will be pulled when finishing away from catching binding reagents measuring, even and analyte be present in the sample and also can not be detected.
Method of the present invention and box body are through following this problem that solved, and its combination that executed in parallel is different in same equipment is measured, and for example sandwich assay is wherein only used one type magnetic mark spike binding reagents (typical case is an antibody) in each is measured.Typical embodiment relates to box body and method; Wherein use four kinds of different antibody to be used for the detection of analyte; Every kind of antibody is to different epi-positions; Wherein two kinds of antibody are as the capture antibody that is fixed in each of two reaction chambers, and two kinds of antibody are as the magnetic mark tracer antibody, and wherein each reaction chamber comprises one of two kinds of capture antibodies.This idea can be promoted, as long as:
At least a binding reagents of catching of-use, and,
-in box body, exist to have the zone of separating at least of catching binding reagents, and (as the reaction chamber of two separations or as a reaction chamber with separated region),
At least two kinds of magnetic mark spike binding reagents of-use; Wherein in each zone, only use one type magnetic mark binding reagents, thereby for example analyte molecule can be attached to the antibody with magnetic mark thing via two different epi-positions never as described in Figure 2.
Therefore the most in the broadest sense; First aspect of the present invention relates to a kind of box body (1) that is used for utilizing in combination mensuration magnetic mark thing (3) check and analysis thing (2); Said box body comprises at least a binding reagents (4) of catching to the binding site of said analyte; And comprise at least two kinds of magnetic mark spike binding reagents (51 and 52) to the other different binding site of said analyte; It is characterized in that said box body comprises at least two reaction chambers (61 and 62); Wherein first reaction chamber (61) comprises and saidly at least aly catches the binding reagents (4) and the first magnetic mark tracer (51), and wherein second reaction chamber (62) comprises the said at least a binding reagents (4) and the second magnetic mark spike binding reagents (52) of catching.This first area (61) does not comprise the magnetic mark spike binding reagents of non-this first magnetic mark spike binding reagents (51), and this second area (62) does not comprise the magnetic mark spike binding reagents of non-this second magnetic mark spike binding reagents (52).
Thereby when creating the interaction of the analyte molecule of having avoided causing the bridge joint compound of not expecting described like Fig. 2 in special zone and two kinds of spike binding reagents (51) and (52), the box body of description and method can be carried out in one and identical reaction chamber in the present invention.This illustrates in the example of Fig. 3, wherein applied magnetic mark spike binding reagents and it is dry on the separated region of box body.As long as liquid gets into this box body and magnetic-particle suspends again, thus so applying a magnetic field keep this magnetic-particle fixing or handle this magnetic-particle on basic vertical direction (by the indication of the arrow z among Fig. 3) towards and away from box body in catch binding reagents on the opposite side.The vertical manipulation of magnetic-particle has avoided magnetic mark spike binding reagents (51) and (52) to contact with each other; And the analyte of having avoided being attached to magnetic mark spike binding reagents (51) can additionally be attached to magnetic mark spike binding reagents (52), and vice versa.
In specific embodiment, said zone (61) and (62) are independent reaction chambers.
In another specific embodiment, said binding reagents is an antibody.
The further details of this box body will be described with embodiment; Wherein this box body comprises two reaction chambers (61 and 62); It has the two kinds of capture antibodies (41 and 42) to two different epi-positions of analyte; And two kinds of tracer antibodies (51 and 52), said two kinds of tracer antibodies have the magnetic mark thing to two other epi-positions on the said analyte.
Use well known in the prior art being used for that protein is fixed to the technology such as plastics, glass, pottery and metallic surface, capture antibody is fixed to the surface of reaction chamber.In specific embodiment, for example can using, the responsive coupling agent of mercaptan comes the combination between reversal reaction surface and the capture antibody.
This box body also comprises the two kinds of magnetic mark tracer antibodies (51 and 52) to two other different epi-positions of said analyte.The method that in the prior art protein is combined with magnetic-particle is well-known.Usually, a magnetic-particle will carry different antibody molecules on its surface, be attached to the quantity of the antibody of single particle but can adjust reaction conditions with control, with the ratio of an antibody of the every particle of final acquisition.Described dissimilar magnetic-particles in the prior art, its shape is different with material.To method described in the invention, typically use to have between about 50 to 2000nm, specifically between about 250 to 750nm, and more specifically be the magnetic-particle of the diameter of about 500nm.This magnetic-particle is used for the tracer antibody that manipulation is attached in magnetic field in first instance.Randomly, the magnetic characteristic of particle also can be used to detect this particle.Alternatively, the detection of magnetic-particle is based on optical characteristics, and for example the size of particle itself (for example, in FTIR (frustrated total internal reflection), measuring) is perhaps through adding chromophoric group (the fluorescence granules of polystyrene that for example, comprises magnetic material) in magnetic-particle.
This box body comprises two reaction chambers (61 and 62) of the detection that is used for analyte (2).Yet this box body can comprise the other reaction chamber of the detection that is used for other analytes.This first reaction chamber (61) comprises said two kinds of capture antibodies (41 and 42) of the different epi-positions that are directed against analyte and is attached to the first magnetic mark tracer antibody (51) of (the 3rd) the in addition epi-position on the antibody.This second reaction chamber (62) comprises above-mentioned two kinds of capture antibodies (41 and 42) and is attached to the second magnetic mark tracer antibody (52) of (the 4th) the again in addition epi-position on the antibody.
In specific embodiment, before using box body, this tracer antibody is applied in the reaction chamber.For example, with this tracer antibody be applied to reaction chamber away from the buffer zone (buffer) of the part of capture antibody and be dried.When sample liquid being introduced in the reaction buffer zone, this tracer antibody dissolving and quilt disperse again.
In optional embodiment, before sample got into reaction chamber, this tracer antibody contacted with sample.This takes place when typically being to supply tracer antibody in the passage between sample inlet and the reaction chamber.It is obvious that in this configuration, thereby be merely able to move in the reaction chamber with the tracer antibody that mode like this designs one type in this passage.When having the sample admission passage of analyte; This tracer antibody is with dissolved; Then with as getting into reaction chamber, perhaps all combined or do not existed under the situation of analyte to get into reaction chamber as tracer antibody at all analytes with the compound of analyte.
In the box body of above description, all to be combined with the bridge joint of two kinds of different antibodies of magnetic-particle compound with analyte and each can not take place.Analyte molecule will only carry a kind of magnetic-particle.
The equipment of describing in the present invention can be applicable to the analyte that needs use any kind of different spike binding reagents with method.The different spike binding reagents of the needs for example reason of antibody possibly be different; And comprise that translating the back (for example modifies; Phosphorylation, glycosylation) in the degraded of difference, proteolysis or processing, form and the conformational change of analyte epi-position with the compound that stops spike binding reagents such as antibody to be attached to other compositions of analyte.
In specific embodiment, this analyte is a troponin.This protein is oligomeric protein, and it comprises on the one hand and form different compounds, and this compound can hide epi-position with respect to antibody, and on the other hand after collecting blood sample this protein be easy to proteolysis and degrade.Cardiac troponin-I in the blood (cTnI) and cardiac troponin-T (cTnT) are the dead indications of heart cell, and are used for the diagnosis of acute myocardial infarction (AMI), unstable angina pectoris, postoperative myocardial damage and the other diseases relevant with the cardiac muscle damage.
The structure of cardiac troponin-I (cTnI) has been shown among Fig. 7.The center section of this molecule (AA30-109) is very stable, but has the possibility that autoantibody is attached to this molecule.The heparin (anti-coagulants) that other sites of some of this molecule can be able to be present in the blood sample hides.The outermost area of this molecule (AA 1-29 and AA 110-209) illustrates less interference, but it tends to more unstable and is cut off in some cases.The difference of the state of oxidation of the difference of the phosphorylation of Ser22 and Ser23 or Cys80 and Cys97 can further influence antibodies.All these can stop tracer antibody to be attached on the particle to the modification of TcnI analyte; And thereby the assay that leads to errors; Yet; In one and identical reaction chamber, use to be attached to the different tracer antibodies that troponin combines and to produce the bridge joint problem of above description, and stop and cause the analyte of false negative result and combining of capture antibody with magnetic-particle.In the reaction chamber of separating, use different magnetic mark tracer antibodies to solve this problem.
Another aspect of the present invention relates to a kind of magnetic mark spike binding reagents that uses with combining to measure the method that detects the analyte (2) in the sample.This method comprises step:
-a) have at least two zones box body (1) of (61 and 62) is provided, wherein will be fixed on the surface of said zone (61 and 62) to the binding reagents (4) of catching of the binding site of said analyte,
-b) compound of the analyte and the first magnetic mark spike binding reagents being provided in said first area (61), this first magnetic mark spike binding reagents is to the other binding site of said analyte (51),
-c) compound of the analyte and the second magnetic mark spike binding reagents (52) being provided in said second area (62), this second magnetic mark spike binding reagents (52) is to the other again binding site of said analyte,
-d) allow step b) and c) in compound combine with the binding reagents (4) of catching in the said zone,
-e) detect step b) and c) in compound catch the combination of binding reagents to this; Wherein this first area (61) do not comprise the magnetic mark spike binding reagents of non-this first magnetic mark spike binding reagents (51) in said method, and this second area (62) does not comprise the magnetic mark spike binding reagents of non-this second magnetic mark spike binding reagents (52).
In specific embodiment, said zone (61) and (62) are reaction chambers.
At binding reagents described in another specific embodiment is antibody.
As above illustrated, will describe this method in detail to the embodiment that wherein uses two kinds of different capture antibodies (41 and 42) and two kinds of different magnetic mark tracer antibodies (51 and 52) to box body.This method may further comprise the steps:
-box body (1) with two reaction chambers (61 and 62) is provided, wherein will be fixed on the surface of said reaction chamber (61 and 62) to two kinds of capture antibodies (41 and 42) of two different epi-positions of said analyte.The details of this reaction chamber has been described hereinbefore.It is well-known in the prior art antibody being used and is fixed on lip-deep method.
-compound of the analyte and the first magnetic mark tracer antibody (51) is provided in said first reaction chamber (61), and the compound of the analyte and the second magnetic mark tracer antibody (52) is provided in said second reaction chamber (62).This compound can form outside reaction chamber, for example when handling sample or in through the fluid passage between sample application and reaction chamber, this tracer antibody is provided.In specific embodiment, within reaction, using tracer antibody during the manufacturing of box body, thereby when sample fluid is introduced reaction chamber, can form the compound with analyte.Be convenient to the formation of this compound through the magnetic actuation of particle.
-allow step b) and c) in compound combine with capture antibody in the said reaction chamber.Between above-mentioned period of energization or after, magnetic-particle is handled towards surface that capture antibody was positioned at.Last in this integrating step applies magnetic field to remove unconjugated tracer antibody away from the direction of this capture antibody.
-detection tracer antibody and analyte are to the combination of capture antibody.This detection can be accomplished through the detected characteristic of measuring particle; For example detect the magnetic of the electrical characteristics of magnetic material, use for example light scattering of optical technology, more specifically FTIR; Detect the existence of particle; Perhaps detect the existence of chromophoric group (for example, fluorescence), this chromophoric group can be present within this magnetic-particle.This detection typically takes place on the surface of fixed trapped antibody within the reaction chamber.Yet, optional embodiment imagination the collected outside of reaction chamber with measure magnetic-particle (for example degenerative condition or wherein for example capture antibody be attached to after the surface relates to the situation that disulfide bond reduces).
Can adjust above method to comprise to sandwich assay, microfluidic device, actuating method; And the modification of detection method; And has a collateral condition: in the method according to the invention; First area (61) does not comprise the second magnetic mark tracer antibody (52), and wherein second area (62) does not comprise the first magnetic mark tracer antibody (51).
Enforcement in biology sensor can be obvious as far as the user, can use algorithm to make up from the signal in two chambers or the zone, and can only net result be shown to the user.
Other layouts of the system and method that the present invention is specialized will be conspicuous to those skilled in the art.
Though will be appreciated that in this article to equipment according to the present invention and discussed preferred embodiment, particular configuration and configuration and material, can in form and details, make various variations or modification and do not depart from the scope of the present invention and spiritual.
Example
The detection of example 1. cardiac muscle troponin Is (cTnI)
Below illustrate the principle of the method for describing in the present invention, wherein use to two kinds of antibody (Ab-S1 (51) and AbS2 (41)) of stabilized zone [S] and the two kinds of antibody (US-Ab1 (52) and US-Ab2 (42)) that are directed against the unstable region [US] of this molecule to the situation of cTnI.In each reaction chamber, only there is the tracer antibody of an epi-position that is attached to this target molecule, thereby the compound of analyte molecule and two kinds of magnetic-particles can not be taken place.Owing on the surface of sensor, have steady component that is directed against molecule and the capture antibody that is directed against the l fraction of molecule, so this mensuration still has the characteristic of robust.Under the situation that has autoantibody (it blocks the binding site of the steady component of this molecule), the particle in the chamber still can be incorporated into the US-Ab2 (42) on the sensor surface.Can't obtain still can to combine the tracer antibody in the chamber under the situation than unstable region of molecule.In the form that Fig. 5 described, reaction surface comprises the combination of two kinds of capture antibody types.
The volume (about 20 μ l) of the blood sample that in the magnetic mark thing is measured, uses is enough greatly sample is divided in 2 chambers and this mensuration is carried out in measure extraly.
In different chamber can with have carry out to other magnetic-particles of the antibody of other targets multiplexing because the bridge joint between those particles is impossible.
The various antibody that are used for the C Troponin I can obtain from the Hytest of for example Finland is commercial.The combination of two kinds of capture antibodies and two kinds of tracer antibodies is described by Hytest, and comprises following combination.
Numeral in the subscript refers to the epi-position of cTnI.
Claims (15)
1. one kind is used for combining to measure the box body (1) that utilizes magnetic mark thing (3) check and analysis thing (2); Said box body comprises at least a binding reagents (4) of catching to the binding site on the said analyte; And comprise at least two kinds of magnetic mark spike binding reagents (51 and 52) of the other different binding site on the said analyte, it is characterized in that said box body comprises at least two zones (61 and 62); Wherein, First area (61) comprises saidly at least aly catches the binding reagents (4) and the first magnetic mark spike binding reagents (51), and wherein, second area (62) comprises at least a binding reagents (4) and the second magnetic mark spike binding reagents (52 of catching;) wherein; Said first area (61) does not comprise the magnetic mark spike binding reagents of the non-said first magnetic mark spike binding reagents (51), and wherein, said second area (62) does not comprise the magnetic mark spike binding reagents of the non-said second magnetic mark spike binding reagents (52).
2. box body as claimed in claim 1; Comprise that two kinds to two different binding sites on the said analyte are caught binding reagents (41 and 42); Wherein, Said first area (61) comprises said first and catches binding reagents (41) and the said first spike binding reagents (51), and wherein, said second area (62) comprises said second and catches binding reagents (42) and the said second spike binding reagents (52).
3. according to claim 1 or claim 2 box body; Comprise that two kinds to two different binding sites on the said analyte are caught binding reagents (41 and 42); And comprise that wherein, said box body comprises two zones (61 and 62) to two kinds of magnetic mark spike binding reagents (51 and 52) of two other the different binding site on the said analyte; Wherein, First area (61) comprises that said first and second catch binding reagents (41 and 42) and the said first magnetic mark spike binding reagents (51), and wherein, said second area (62) comprises that said first and second catch binding reagents (41 and 42) and the said second magnetic mark spike binding reagents (52).
4. like each described box body among the claim 1-3, wherein, said binding reagents is an antibody.
5. like each described box body among the claim 1-4, wherein, said zone is a reaction chamber.
6. like each described box body among the claim 1-5, wherein, said analyte is a troponin.
7. like each described box body among the claim 1-6, wherein, said analyte is cardiac muscle troponin I (cTnI).
8. one kind is used the utilization of magnetic mark spike binding reagents to combine to measure the method that detects the analyte (2) in the sample, and said method comprises step:
-a) have at least two zones box body (2) of (61 and 62) is provided, wherein, will be fixed on the surface of said zone (61 and 62) to the binding reagents (4) of catching of the binding site on the said analyte,
-b) compound of the said analyte and the first magnetic mark spike binding reagents (51) being provided in said first area (61), the said first magnetic mark spike binding reagents is to the other binding site on the said analyte,
-c) compound of the said analyte and the second magnetic mark spike binding reagents (52) being provided in said second area (62), the said second magnetic mark spike binding reagents is to the other again binding site on the said analyte,
-d) allow step b) and c) in said compound combine with the said binding reagents of catching in the said zone,
-e) detect step b) and c) in said compound to said combination of catching binding reagents; Wherein, The magnetic mark spike binding reagents that does not comprise the non-said first magnetic mark spike binding reagents (51) in first area described in the said method (61), and said second area (62) does not comprise the magnetic mark spike binding reagents of the non-said second magnetic mark spike binding reagents (52).
9. method as claimed in claim 8, wherein, said binding reagents is an antibody.
10. like claim 8 or 9 described methods, wherein, said zone is a reaction chamber.
11. like each described method among the claim 8-10, wherein, said analyte is a troponin.
12. like each described method among the claim 8-11, wherein, said analyte is cardiac muscle troponin I (cTnI), and wherein, tracer antibody (41) and capture antibody (51) are attached to the different epi-positions on the stabilized zone of cTnI, and,
Wherein, tracer antibody (42) and capture antibody (52) are attached to the different epi-positions on the unstable region of troponin.
13. one kind is used to use magnetic actuation to carry out the equipment that combines mensuration, comprises box body as claimed in claim 1.
14. kit; At least three kinds of different binding reagents that comprise the different binding sites that are attached on the same analyte wherein, are used the magnetic-particle mark at least two kinds in the said binding reagents; And wherein, at least a in the said binding reagents without the magnetic-particle mark.
15. kit as claimed in claim 14, wherein, said analyte is a cardiac troponin, and wherein, the first unmarked antibody and the first magnetic mark antibodies different epi-positions to the stabilized zone of troponin, and,
Wherein, the second unmarked antibody and the second magnetic mark antibodies different epi-positions to the unstable region of troponin.
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| EP09305887 | 2009-09-23 | ||
| PCT/IB2010/054094 WO2011036597A1 (en) | 2009-09-23 | 2010-09-10 | Binding assay with multiple magnetically labelled tracer binding agents. |
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| CN102597774B CN102597774B (en) | 2015-05-13 |
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| US (1) | US10041939B2 (en) |
| EP (1) | EP2480887B1 (en) |
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- 2010-09-10 EP EP10760077.7A patent/EP2480887B1/en active Active
- 2010-09-10 US US13/497,813 patent/US10041939B2/en active Active
- 2010-09-10 WO PCT/IB2010/054094 patent/WO2011036597A1/en active Application Filing
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| CN103776995A (en) * | 2012-08-21 | 2014-05-07 | 阿斯特里姆有限责任公司 | Method for performing a biochemical analysis, in particular in space |
| US9921214B2 (en) | 2012-08-21 | 2018-03-20 | Airbus Ds Gmbh | Method for performing a biochemical analysis, especially in outer space |
| CN105158483A (en) * | 2015-09-16 | 2015-12-16 | 北京九强生物技术股份有限公司 | Quantitative determination kit for hypersensitivity of Troponin I and detection method |
| CN109580954A (en) * | 2015-09-16 | 2019-04-05 | 北京九强生物技术股份有限公司 | A kind of super quick quantitative determination reagent kit and its detection method of human troponin I |
| CN109580954B (en) * | 2015-09-16 | 2022-03-22 | 北京九强生物技术股份有限公司 | Hypersensitivity quantitative determination kit for human troponin I and detection method thereof |
| CN107356758A (en) * | 2017-08-25 | 2017-11-17 | 李红俊 | A kind of lung cancer detection kit and its application method |
| CN107356758B (en) * | 2017-08-25 | 2019-11-22 | 李红俊 | A kind of lung cancer detection kit and its application method |
| CN109613242A (en) * | 2018-11-01 | 2019-04-12 | 深圳天辰医疗科技有限公司 | A kind of creatine kinase isozyme detection kit and preparation method thereof |
| CN112710855A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | BNP detection kit and BNP detection method |
Also Published As
| Publication number | Publication date |
|---|---|
| EP2480887B1 (en) | 2015-12-16 |
| US20120178186A1 (en) | 2012-07-12 |
| WO2011036597A1 (en) | 2011-03-31 |
| EP2480887A1 (en) | 2012-08-01 |
| CN102597774B (en) | 2015-05-13 |
| US10041939B2 (en) | 2018-08-07 |
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